Adequate supervision of the safety of industrial products depends heavily on the detection of microbial contamination of the product. This microbial contamination can exist in a wide variety of industrial products including food, drinking water and health and beauty aids. A normal approach to detecting microbial contamination involves tests which depend upon the incubation of a sample taken from the product in a media which is suitable for the growth of micro-organisms. This approach involves the growth of microbes to ensure their viability and at the same time the multiplication of signals in order to simplify their detection. However, in many instances it requires several days to perform the test which imposes severe delays in manufacturing and inventory cost This is extremely critical in cases where the product is labile and the result of microbial testing is a shorter shelf life. Evidently then it is extremely important to develop ways of performing the microbial testing in a rapid manner.
Rapid testing methods which detect microbes without requiring a multiplication by growth usually involve labels which have been developed to effectively mark any viable micro-organism through the use of luminescence or fluorescence.
The general drawback with these methods has been the limit placed on the entire operation by the effectiveness of the instrumentation which must be capable of picking out the labeled microbe from other interfering signals with sufficient reliability to be useful in everyday practice. Practical use of such systems requires the ability to have a sensitivity to contamination which is very high detecting 100 or less microbes per milliliter of product. There is also a simultaneous requirement to have an extremely low false alarm rate of less than 1% for example.
One currently used instrumentation which has been attempted as a solution is a fluorescent flow cytometer wherein a diluted sample passes through a laser beam and photodetectors are used to note any fluorescence. Such a device when coupled with a prior device for fluorescently labeling each individual viable microbe, appears to be a useful tool in this area of microbial contamination detection. However, in spite of many attempts, this technology has not proved practical for a wide class of industrial products primarily due to the limitations in the sensitivity and/or specificity which arises.
The above discussed instrumentation and fluorescent labeling generally falls into two categories or two approaches to labeling the contaminating microbes. They both depend upon the action of a ubiquitous enzyme within the microbe-organism to create an optical signal. In one instance the resultant is a luminescent reaction while in the other instance the microbe is rendered fluorescent. The applicable use for either of these labeling method is limited by either the sensitivity of the luminescence method, so that enough light is not generated by a single microbe to be detected, or the specificity with regard to the fluorescent methods wherein any light from the labeled microbe cannot be distinguished from background fluorescent sources.
The automation of fluorescent methods of rapid microbiology yields two currently used approaches. In the fluorescent flow cytometry approach, a diluted suspension of a product to be tested is interrogated by passing it through a laser spot and detecting the resultant fluorescence of labeled microbes. On the other hand in the method known as the solid phase cytometry for instance as described in U.S. Pat. No. 5,663,057, a sample of a liquid product is passed through a membrane filter with sufficiently small pore size to retain any microbes and the filter is subsequently scanned by laser beam to detect any labeled microbes.
These two methods use different sampling means and address different products. For example some samples may not be filterable and thus cannot be used with the solid phase cytometer. Furthermore the level of performance which measures the sensitivity to the contaminating element which is obtained from the fluorescent flow cytometry is different from the solid phase cytometry. In fact, the solid phase cytometry is consistently more effective at detecting contamination. This difference is not due to the relative sensitivity but instead is due to the relative specificity. That is, both detectors have sufficient sensitivity to respond to a single microbe but the solid phase cytometer uses a set of sophisticated discriminators which are applied to a digitized waveform representing the fluorescent signal and these discriminators are based on the relative amplitude and detailed phase shape of individual fluorescent signals obtained at two or more optical wavelengths. It is for this reason that the solid phase cytometer is more effective at detecting contamination than the currently used fluorescent flow cytometers.
This difference in discrimination ability occurs because, when contrasted with the solid phase cytometers, the commercial fluorescent flow cytometer employ analog circuits which produce the feature values of the pulse waveform resulting from the particle fluorescence. This use of analog pulse processing limits the features which can be measured to their pulse integral, pulse height and pulse width. Thus, a significant amount of relevant information concerning the shape of the waveform is lost.
However it must be pointed out that, although digital processing has been applied to flow cytometers, its use has generally been limited by data processing speed. That is, when the sampling rate is made sufficiently high to obtain the required resolution for analyzing a single pulse, the processing system cannot keep up with the random pulse arrival rate. That is, the lowest sampling rate still produces an output for which continuous sampling is not possible. In order to resolve this issue a compromise in digital resolution is usually used and thus a compromise in the potential performance.
Yet another approach to obtaining additional information regarding the variation in fluorescent signals over time has been to use an array of detectors (linear CCD) which extend along the particle trajectories. In this method, the signal from each detector is processed in an analog manner and the results are combined to obtain a signal waveform. While this signal has been shown useful to measure the fluorescent decay it is a complicated system which must be precisely set up and it is limited by the relative sensitivity of adjacent detectors.